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Definitions

• This invention relates to the inhibition of blood coagulation, especially during organ rejection.
• Xenografting is currently hindered, however, by the severe and well-documented problems of rejection. This process can be divided into distinct stages, the first of which occurs within minutes of transplantation. This is known as the hyperacute response and is caused by existing antibodies in the recipient which recognise and react with foreign antigens on the endothelial cells (ECs) of the xenograft. This recognition triggers the complement cascade which in turn leads to lysis and death of ECs of the transplant.
• ECs endothelial cells
• This initial hyperacute rejection is then reinforced by the delayed vascular response (also known as acute vascular rejection or delayed xenograft rejection).
• delayed vascular response also known as acute vascular rejection or delayed xenograft rejection.
• the lysis and death of ECs during the hyperacute response is accompanied by oedema and the exposure of adventitial cells, which constitutively express tissue factor (TF) on their surface.
• TF tissue factor
• Tissue factor is thought to be pivotal in the initiation of the in vivo coagulation cascade, and its exposure to plasma triggers the clotting reactions.
• Thrombin and TNF- ⁇ become localised around the damaged tissue and this induces further synthesis and expression of TF by ECs.
• XNAs xenoreactive natural antibodies
• vascularised regions of the xenograft thus become sites of blood clots, a characteristic of damaged tissue. Blood flow is impaired and the transplanted organ becomes ischaemic. A fuller account of delayed vascular rejection can be found in Bach et al. (1996).
• xenogeneic organs in transplants is therefore hindered by an initial hyperacute rejection followed by a prolonged vascular rejection, possibly followed by T-cell mediated rejection. Inhibition of the mechanisms responsible for these rejections could facilitate the use of xenografts.
• inhibitors are not a particularly suitable approach. Completely inhibiting complement in a recipient animal is tantamount to immunosuppression, leaving the subject prone to opportunistic infections. Similarly, inhibiting the coagulation cascade in a recipient will leave the animal susceptible to uncontrolled post-operative bleeding. Therefore the inhibitors should desirably be localised in the recipient to the site of the xenograft.
• Transgenic pigs expressing human DAF and/or CD59 have been reported (McCurry, 1996). Cardiac rejection took twice as long to occur with the transgenic xenografts than with controls.
• tissue factor pathway inhibitor is known to inhibit the function of the active complex which is normally formed between tissue factor, factor Vila, and factor Xa.
• TFPI tissue factor pathway inhibitor
• TFPI tissue factor pathway inhibitor
• Kunitz domain I is responsible for binding tissue factor and factor Vila
• domain II binds factor Xa
• Tick anticoagulant peptide TMP is a specific and potent inhibitor of factor Xa. This 60 amino acid polypeptide has been purified from the soft tick Ornithodoros moubata.
• snake venoms also contain anticoagulant polypeptides.
• anticoagulant polypeptides For instance, a 231 amino acid protein C activator has been purified from the venom of the snake Agkistrodon contortrix contortrix (McMullen, 1989; Kisiel, 1987).
• Hirudin is the anticoagulant protein utilised by the leech Hirudo medicinalis when extracting blood from its victim. It is highly potent and binds to thrombin at a 1 :1 ratio with a dissociation constant in the femtomolar range. The active site of thrombin is masked in the stable complex and so the hirudin prevents fibrinogen breakdown, thus inhibiting clot formation.
• P-selectin also known as CD62
• CD62 P-selectin
• the rejection might be xenogeneic or allogeneic.
• a protein comprising a region with anticoagulant activity and a region which can anchor said protein to a cell membrane.
• this is a chimeric protein, that is to say the anchor region and anticoagulant region are derived from different proteins.
• the anticoagulant region can comprise the sequence of any anticoagulant polypeptide.
• anticoagulant polypeptides include heparin, TAPs, antithrombin, hirudins, and TFPIs, along with their functional derivatives, such as fragments and derivatives which retain anticoagulant activity.
• Anticoagulant derivatives of thrombin, normally a procoagulant, have also been reported (Dang, 1997).
• the anticoagulant region comprises the sequence of a hirudin.
• Hirudins include hirudin, hirudin derivatives, analogs ("hirulogs"), and variants (eg. hirudisins). For instance, it has been reported that sulphation at Tyr-64 increases the anticoagulant activity of hirudin, and that hirudisin-2 is a more potent inhibitor of thrombin activity than hirudin itself (eg. Knapp, 1992; Skern, 1990).
• the anticoagulant region might comprise the sequence of a tissue factor pathway inhibitor (TFPI).
• TFPIs include TFPI itself and derivatives or analogs thereof which retain inhibitory activity.
• the TFPI sequence comprises Kunitz domains I and II of TFPI itself.
• the anticoagulant region might comprise the sequence of a tick anticoagulant peptide (TAP).
• TAPs include TAP itself and derivatives or analogs thereof which retain inhibitory activity. For instance, the potency of FXa inhibition by TAP has been enhanced by site-directed mutagenesis (eg. Mao, 1995).
• anticoagulant regions could, for instance, comprise the sequence of a protein C activator, such as those isolated from snake venom (eg. McMuUen, 1989; Kisiel, 1987), or the sequence of anticoagulants isolated from snake venoms which act other than via protein C activation, or their derivatives or analogs which retain anticoagulant activity.
• a protein C activator such as those isolated from snake venom (eg. McMuUen, 1989; Kisiel, 1987)
• anticoagulants isolated from snake venoms which act other than via protein C activation, or their derivatives or analogs which retain anticoagulant activity.
• the anchor region can be any entity which can attach the protein to a cell membrane. Suitable examples include transmembrane sequences from membrane proteins and GPI anchors. Preferably the anchor region is a sequence capable of attaching the protein to a lipid bilayer, such as the transmembrane regions of the HLA class I or CD4 proteins. It may also be desirable for the protein to comprise the cytoplasmic domain which is usually associated with said transmembrane regions, such as the CD4 cytoplasmic domain, and/or the extracellular domains immediately juxtaposed with the cell membrane, such as CD4 domains 3 and 4. Alternatively the anchor region might be a sequence conferring on the protein the ability to associate extracellularly with a membrane protein without the protein itself being inserted into the cell membrane.
• a protein according to the first aspect further comprising a targeting sequence which prevents the protein from being constitutively expressed at the cell surface.
• the targeting sequence is a polypeptide sequence which can target a nascent polypeptide to a secretory granule, and more preferably the secretory granule is one which does not fuse with the cell's plasma membrane until the cell is suitably stimulated.
• the secretory granule fuses with the plasma membrane during EC activation which occurs during organ rejection.
• the targeting sequence is preferably one which targets a nascent polypeptide to a Weibel- Palade body, such as the relevant sequence from P-selectin.
• the protein according to the second aspect of the invention comprises an anticoagulant sequence and the transmembrane and cytoplasmic domains of P-selectin. The domains from P-selectin thus provide both the anchor sequence and the targeting sequence.
• a polynucleotide encoding a protein according to the present invention.
• the polynucleotide is DNA.
• the polynucleotide comprises sequences suitable for the regulation of expression of protein according to the invention.
• This expression can preferably be controlled, such as cell-specific control, inducible control, or temporal control.
• expression might be specific for ECs, or might be regulated in response to cell activation.
• a vector comprising a polynucleotide according to the third aspect.
• the term "vector” signifies a molecule which is capable of transferring a polynucleotide to a host cell.
• the vector is a DNA vector and, more preferably, is capable of expressing R A encoding a protein according to the invention. Numerous suitable vectors are known in the art.
• the vector is suitable for the production of a transgenic animal.
• Vectors suitable for the generation of transgenic pigs are described in Heckl-Ostreicher (1995), McCurry (1996), White (1995), Yannoutsos (1995), and Langford (1996).
• Minigene vectors suitable for the generation of transgenic mice are described in Diamond (1995).
• a delivery system comprising a molecule of the first, second, third, or fourth aspects and means to deliver said molecule to a target cell.
• vectors according to the fourth aspect may also function as suitable delivery systems.
• certain delivery systems according to this fifth aspect may also inherently be vectors, but this is not always the case.
• a viral vector can also function as a delivery system, whereas a liposomal delivery system is not a vector.
• the delivery system may be viral or non- viral.
• Non- viral systems such as liposomes, avoid some of the difficulties associated with virus-based systems, such as the expense of scaled production, poor persistence of expression, and concerns about safety.
• the delivery system is suitable for use in gene therapy. Numerous appropriate delivery systems are known in the art.
• the delivery system will be targeted so that molecules according to the present invention are taken up by cells suitable for transplantation, or cells which have been transplanted. More preferably the delivery system will be specific for these cells.
• the delivery system may be targeted to a specific organ, such as the heart or the kidney, or to a specific cell type, such as endothelial cells.
• the delivery system may, for example, be a receptor-mediated delivery system, being targeted to receptors found on target cells.
• the delivery system may be targeted to receptors found on heart cells, preferably to receptors found exclusively on heart cells, or it may be targeted to receptors found on endothelial cells, preferably to receptors found exclusively on endothelial cells, or to receptors found on activated endothelial cells, such as E-selectin or P-selectin.
• the delivery system is preferably suitable for the generation of transgenic animals.
• the delivery system may be targeted to a gamete, a zygote, or an embryonic stem cell.
• a method of transfecting a cell with a vector according to the invention may involve the use of a delivery system according to the invention.
• the cell type is not restricted and may be prokaryotic or eukaryotic. Transfection can occur in vivo or ex vivo.
• the cell is preferably eukaryotic, more preferably an endothelial cell.
• porcine endothelial cells for example, is described in Heckl-Ostreicher (1995).
• the cell is suitable for the generation of a transgenic animal. More preferably, the cell is a gamete, a zygote, or an embryonic stem cell.
• the transfection of murine ova by microinjection to generate transgenic mice, for example, is described in Diamond (1995), and the microinjection of porcine zygotes, for instance, to generate transgenic pigs is described in Yannoutsos (1995), Langford (1996), and White (1995).
• a seventh aspect of the invention there is provided a cell transfected according to the sixth aspect.
• the cell is preferably able to express two or more different proteins according to the invention, each of which inhibits the coagulation cascade at a different stage.
• the anticoagulant region in one protein might comprise a TFPI, whilst in the other it comprises a hirudin.
• biological tissue comprising a cell according to the invention.
• biological tissue includes collections of cells, tissues, and organs. Accordingly the definition includes, for example, fibroblasts, a cornea, nervous tissue, a heart, a liver, or a kidney.
• an animal comprising a cell and/or biological tissue according to the invention.
• the animal is suitable for the production of organs for transplantation into humans.
• the animal is a mammal, and more preferably it is a transgenic pig or a transgenic sheep.
• the animal might be treated whilst alive such that it comprises transgenic biological tissue (ie. treated by gene therapy).
• a live animal is transfected with a vector according to the invention in order to produce a transgenic animal.
• a vector according to the invention could be specifically delivered to endothelial cells in a pig to produce transgenic organs suitable for xenotransplantation.
• the animal might be born as a transgenic animal.
• Various suitable approaches for generating such transgenic animals are known in the art (eg. Bradley & Liu, 1996; Clarke, 1996; Wheeler, 1994).
• direct manipulation of the zygote or early embryo, by microinjection of DNA for instance is well known, as is the in vitro manipulation of pluripotent cells such as embryonic stem cells.
• Retroviral infection of early embryos has proved successful in a range of species, and adenoviral infection of zona-free eggs has been reported.
• Transgenesis and cloning of sheep by nuclear transfer has also been described (eg. WO97/07668).
• a method of rendering biological tissue suitable for transplantation comprising expressing one or more proteins according to the present invention in said biological tissue, preferably in its endothelial cells.
• the biological tissue may be so rendered either in vivo or ex vivo.
• an animal organ may be in vivo transfected with a vector according to the invention, or an organ could be transfected ex vivo before transplantation or in vivo after transplantation.
• a method of transplantation comprising transplanting biological tissue according to the invention from a donor animal into a recipient animal.
• the method is for xenotransplantation and the donor biological tissue is xenogeneic with respect to the recipient animal.
• Figure 1 shows maps of hirudin-CD4 chimeric proteins and constructs according to the invention.
• A HLA-hirudin-CD4 constructs with glycine linkers.
• B HLA-hirudin-CD4 construct with human P-selectin C-terminal, with the specific targeting sequence underlined. Transmembrane (TM), stop transfer (ST), and cytoplasmic (C) regions of CD4 are indicated.
• Figure 2 shows FACS profiles for HLA-hirudin-CD4 constructs expressed in DAP.3 fibroblasts.
• Figure 3 shows FACS profiles for HLA-hirudin-CD4-P-selectin cDNA constructs expressed in CHO-K1.
• Figure 4 shows that hirudin-CD4 expressing fibroblasts bind thrombin.
• Figure 5 shows the specificity of thrombin binding to cells expressing hirudin-CD4.
• Figure 6 shows thrombin binding to CHO-K1 cells transfected with HLA-hirudin constructs.
• Figure 7 shows that inactivation of thrombin abolishes thrombin binding to hirudin-CD4 at the cell surface.
• Cells expressing hirudin-G2-CD4 were incubated with thrombin or inactivated thrombin and stained for thrombin binding with anti-prothrombin or anti-thrombin-hirudin antibodies.
• Figure 8 shows maps of TFPI-CD4 chimeric proteins and constructs according to the invention.
• Figure 9 shows flow cytometry profiles of DAP.3 cells expressing TFPI tethered to the cell surface.
• Figure 10 shows specific FXa binding to cell surface bound TFPI ] . 276 -CD4 and TFPI,. 183 -CD4.
• Figure 11 shows the blocking of FXa binding by a polyclonal anti-TFPI immunoglobulin fraction.
• Figure 12 shows the blocking of FXa binding by monoclonal antibodies directed against Kunitz domains I and II.
• Figure 13 shows the inhibition of FXa by cells expressing TFPI._ 276 -CD4 and TFPIj. 183 -CD4.
• the mean time for a FXa-specific chromogenic substrate to reach OD 405 0.1 is shown for transfected DAP.3 cells incubated with FXa. Values for control cells were subtracted and error bars indicate standard deviations.
• Figure 14 shows that an active TF ⁇ FVIIa complex is required for maximal binding to TFPI-CD4 chimeric proteins.
• Figure 15 shows the specificity of thrombin binding to immortalised porcine endothelial cells (IPEC) expressing hirudin-CD4, and also shows the effect of cell-surface hirudin-CD4 expression on clotting times.
• IPEC immortalised porcine endothelial cells
• Figure 16 shows the distribution of ACTH and hirudin in D 16/16 cells, as revealed by fluorescence.
• Figure 17 shows the change in cellular distribution of hirudin-CD4-P-selectin after PMA stimulation
• Figure 18 shows that TFPI-CD4 expressed on IPEC retains its binding properties.
• Figure 19 shows the competitive binding of porcine and human tissue factors.
• Figure 20 shows that TFPI-CD4 prolongs clotting times when expressed on IPEC surface.
• Figure 21 shows the anti-coagulant effect of co-expression of TFPI-CD4 and hirudin-CD4.
• Hirudin fused with HLA class I signal peptide and linked to domains 3 and 4 of human CD4 is tethered to the cell membrane
• hirudin variant 1 To express heterologous hirudin constructs in mammalian cells, the cDNA for the membrane- targeting signal peptide leader sequence from human HLA class I A2.1, amino acids -1 to -24 (Holmes, 1987), was fused to hirudin variant 1 (Dodt, 1984) using PCR with overlapping extension ( Figure 1).
• the HLA A2.1 leader sequence was amplified using primers:
• the hirudin sequence was amplified using primers:
• the resulting PCR products (108 and 228 bp) were purified by agarose gel electrophoresis and then used in a third PCR using flanking primers hla-1 and hir-3.
• the resulting PCR product (300 bp) was digested with Sail and BamHl and subcloned into pBluescript SK(+) (Stratagene).
• the oligonucleotide pair consisted of 5'-aattaggaggttctggaggctgca-3' ⁇ SEQ ID 5> (containing a mutated EcoRI recognition sequence and a Pstl site) and 5'-gcctccagaacctcct-3' ⁇ S ⁇ Q ID 6>;
• G2 and 3 consisted of the core sequence (GGSGG) repeated two or three times, respectively.
• the glycine linker oligonucleotides were annealed and each ligated into the EcoRI/Pstl site of plasmids containing the HLA-hirudin cassette, prior to the insertion of the CD4 anchor.
• CD4 166 _ 435 was amplified using primers:
• the resulting PCR product was cloned into pBluescript and sequenced.
• V 328 was found to be mutated to A 328 .
• the Pstl/B ⁇ mHl CD4 fragment was subcloned into HLA-hirudin -Gl, -G2, & -G3 plasmids, and these constructs were verified by DNA sequence analysis.
• Each of the three cDNA constructs were subcloned into the B ⁇ mHl site of the mammalian expression vector pH ⁇ Actpr-lgpt (Gunning, 1987), containing the human ⁇ -actin enhancer and promoter region in conjunction with an SV40 enhancer element driving the gpt resistance gene, allowing the selection of clones in the presence of mycophenolic acid ( Figures 1C & ID).
• the orientation of the final constructs was verified by restriction endonuclease mapping.
• Vectors containing the individual HLA-hirudin-Gl/2/3-CD4 constructs were transfected into mouse fibroblast cell line DAP.3 (Marguelies, 1983) with calcium-phosphate according to standard protocols. After 18 hours growth in DMEM medium (Gibco) supplemented with 5% fetal calf serum, ampicillin, streptomycin, and glutamine, cells were glycerol treated for 30 seconds. Cells were then washed twice with phosphate buffered saline (PBS), and new medium including xanthine, hypoxanthine, and mycophenolic acid to a final concentration 12 ⁇ g/ml, was added.
• DMEM medium Gibco
• PBS phosphate buffered saline
• DAP.3 cells transfected with a human class II construct expressing HLA-DR (cell line 531) (Lechler, 1988) grown in identical mycophenolic acid-containing culture medium.
• anticoagulant polypeptides can be stably expressed on the cell surface.
• Hirudin-CD4 with a targeting sequence from the C-terminal of P-selectin is expressed at the cell surface of CHO-K1
• HLA-hirudin-Gl/G2-CD4-P-selectin constructs were subcloned into the BamHl site of pH ⁇ Actpr-lgpt and transfected into CHO-Kl cells (ATCC CCL61), grown in RPMI 1640 medium (Gibco) supplemented with 5% fetal calf serum, ampicillin, streptomycin, and glutamine.
• Transfection was by electroporation according to standard protocols. Briefly, 5x10 6 cells were resuspended in 350 ⁇ l serum-free medium and transferred to a 1 ml electroporation cuvette with a 0.4 cm space between electrodes (Bio-Rad). After addition of 10 ⁇ g plasmid DNA in 150 ⁇ l, samples were gently shaken and kept on ice. Cells were subjected to electroporation at infinite resistance, 960 ⁇ F and 350 V in a Gene Pulser apparatus (Bio-Rad). The day after electroporation, cells were washed twice with PBS and new medium including mycophenolic acid, xanthine, and hypoxanthine was added.
• CHO-Kl cells were transfected with full length human P-selectin (Johnston, 1989), which was subcloned as a 3142bp Sail fragment into pH ⁇ Actpr-lneo containing an SV40-driven neomycin (G418) resistance gene. These cells were treated with 400 ⁇ g/ml G418 and after 2 weeks individual clones were picked with cotton swabs and transferred to 12- well plates. Surviving clones were analysed for hirudin and CD4 expression using 4158-81-7 at 10 ⁇ g/ml and an undiluted OKT-4 hybridoma supernatant.
• chimeric proteins comprising the P-selectin targeting sequence remain functional when expressed at the cell surface.
• Stably transfected cells were grown in T75 culture flasks for 36 hours before each experiment.
• DAP.3 cells were detached using a cell scraper, whilst CHO-Kl cells were detached from the plastic by treatment with PBS, 5 mM EDTA for 10 minutes at 37°C.
• PBS 0.1% BSA
• 2.5xl0 5 cells in 150 ⁇ l were incubated for 1 hour at 37°C with increasing concentrations of thrombin.
• the cells were washed four times with PBS containing 0.1% BSA and further incubated for 30 minutes on ice with rabbit anti-human prothrombin immunoglobulins (lO ⁇ g/ml in lOO ⁇ l) (Dakopatts).
• hirudin expressed at the cell surface retains the ability to bind thrombin and glycine linker length did not influence thrombin binding.
• DAP.3 HLA-hirudin-G3-CD4 transfectants were pre-incubated on ice for 30 minutes with 10 ⁇ g/ml anti-hirudin mAb or appropriate controls (mouse IgGl and IgG2a, Dakopatts) for 30 minutes on ice, and washed twice in PBS containing 0.1% BSA before incubating with thrombin for 1 hour at 37°C as above. Thrombin binding was analysed as above.
• thrombin binding by hirudin-CD4 was demonstrated by incubation with thrombin and comparing labelling with mAb 4107-76-1 (Schlaeppi, 1991) and anti -prothrombin immunoglobulins. 4107-76-1 is directed against the hirudin-thrombin complex and detects neither hirudin without thrombin nor thrombin bound to endogenous thrombin receptors. As shown in Figure 5B, thrombin binding detected with 4107-76-1 paralleled the binding observed with the anti-prothrombin immunoglobulin fraction.
• hirudin expressed on the surface of DAP.3 cells retains specific thrombin binding.
• IPEC Immortalised porcine epithelial cells
• Hirudin expressed on the surface of IPEC thus binds thrombin and also inhibits the clotting of human plasma. 4.
• Hirudin-CD4-P-selectin expressed by CHO-Kl cells binds thrombin
• hirudin-CD4-P-selectin also binds thrombin when expressed at the surface of CHO-Kl cells. These cells were incubated with thrombin for 1 hour at 37°C. After staining with anti-prothrombin immunoglobulins and addition of a second FITC-labelled antibody layer, cells were analysed by flow cytometry.
• results from two CHO-Kl transfectants expressing hirudin-Gl-CD4 and hirudin-G2-CD4 are shown in Figures 6C and 6D. Except for a slightly increased thrombin binding due to better expressed chimeric proteins (higher mfi's), no major differences in binding profiles were detected compared to transfectants expressing hirudin linked to the CD4-P-selectin anchor.
• Hirudin-CD4-P-selectin is stored in secretory granules and can be released on activation
• hirudin-CD4-P-selectin a secretory murine pituitary cell line (D 16/16) was transiently transfected with cDNA encoding either hirudin-CD4-P-selectin or hirudin-CD4.
• This cell line was chosen for two reasons. Firstly, these cells are known to express ACTH in specific storage granules, which are discharged at the cell surface on activation with phorbol esters. Secondly, endothelial cells (which would appear to be the ideal cell type to investigate intracellular targeting of the P-selectin construct) rapidly lose their Weibel-Palade storage granules during in vitro culture.
• hirudin-CD4-P-selectin can be targeted to specific intracellular storage granules, and that functional chimeric molecules can be released and exposed at the cell surface upon activation.
• thrombin 210 nmol in 50 ⁇ l Tris-buffered saline (TBS), 0.1% BSA, pH 7.4 was pre-incubated for 1 hour at 37°C with either:
• hirudin dodecapeptide analog comprising hirudin residues 53-64, with sulfato-Tyr64 (American Diagnostica) at 100-fold molar excess.
• the thrombin-dependent catalytic activity was analysed with a small chromogenic oligopeptide substrate (H-D-Phe-Pip-Arg-pNA-2HCl ("S-2238") (Quadratech). To ascertain whether thrombin was inactivated by PPACK-HC1 and hirudin, 5 ⁇ l of each reaction mixture were diluted with 95 ml TBS, 0.1% BSA and incubated with 50 ⁇ l 4mM S-2238 for 10 minutes at 37°C.
• S-2238 small chromogenic oligopeptide substrate
• TFPI In order to tether TFPI to the cell membrane, a fusion protein consisting of human CD4 16M35 linked either to full length TFPI including all three Kunitz domains (TFPI,_ 276 ) or to a truncated form of TFPI lacking Kunitz domain III and the C-terminal (TFPI H83 ) (Wun, 1988) ( Figure 8).
• TFPI is a mammalian protein and hence contains an endogenous signal peptide.
• CD4 166 _ 435 was amplified as described above.
• the complete TFPI-CD4 cDNAs were ligated into the BamHl site of the pH ⁇ Actpr-lgpt expression vector.
• DAP.3 cells maintained in supplemented DMEM were transfected with calcium- phosphate as above.
• Clones were analysed for TFPI and CD4 expression by FACS using murine anti -human TFPI mAbs 4903 or 4904 (American Diagnostica), both at 10 ⁇ g/ml, and an undiluted OKT-4 hybridoma supernatant (Reinherz, 1979).
• 4903 is directed against Kunitz domain I
• 4904 is directed against Kunitz domain II.
• 10 5 cells for each sample were analysed and, as above, cell line 531 was used as a control.
• both TFPI,. 276 -CD4 and TFPI, .lg3 -CD4 can be expressed at the cell surface.
• TFPI 1 . 183 -CD4 and TFPI,. 276 -CD4 tethered to the cell surface confer FXa binding
• Stably transfected DAP.3 cells were detached by treatment with PBS, 5 mM EDTA for 10 minutes at 37°C. After 4 washes with excess PBS, 0.1 % BSA (w/v), 2.5xl0 5 cells in 100 ⁇ l were incubated for 1 hour at 37°C with increasing concentrations of FXa.
• Cells were incubated on ice for 30 minutes with 4901, 4903, or 4904 at increasing concentrations, using an anti-haemoglobin antiserum (Dakopatts) as a negative control. The cells were then washed twice in PBS, 0.1% BSA, and further incubated with 5 nM FXa for one hour at 37°C. The cells were then washed and incubated with RAFX-IG as above and analysed for FXa binding by FACS.
• Dakopatts anti-haemoglobin antiserum
• TFPI 1 183 -CD4 and TFPI,. 276 -CD4 are both functionally active against FXa
• Transfected DAP.3 cells were detached as described above and washed 4 times with excess TBS, pH 7.4, 0.1% BSA.
• 0.5xl0 6 cells in 100 ⁇ l per well were incubated for 1 hour at 37°C with various concentrations of FXa.
• 50 ⁇ l of 4 mM S-2765 were added and cells were further incubated for 2 hours at 37°C.
• FXa activity was inhibited by expressed TFPI-CD4 in a dose dependent manner with the greatest inhibition noted when low concentrations of FXa (0.16 nM) were added ( Figure 13).
• Figure 13 In a series of experiments, no significant difference in FXa inhibition was observed between cells expressing TFPI, .183 -CD4 or TFPI,. 276 -CD4.
• Kunitz domain II retains its function when tethered to the cell surface in both TFPI,. 183 -CD4 and TFPI,. 276 -CD4.
• Recombinant human and FVIIa were produced in E. coli and CHO-Kl, respectively (O'Brien, 1994). These were mixed in equimolar concentrations and incubated at 25°C for 15 minutes to obtain a TF,. 219 /FVIIa complex.
• DAP.3 cells expressing either TFPI,. 276 -CD4 or TFPI,. 183 -CD4 were incubated with 5 nM FXa for 1 hour at 37°C. Cells were washed twice and TF,. 219 /FVIIa complex was added to 2.5x10 5 cells in 100 ⁇ l. After 1 hour at 37°C transfectants were washed twice and incubated with 50 ⁇ l polyclonal rabbit anti-TF immunoglobulins (2.5 ⁇ g/ml) for 30 minutes on ice followed by 2 washes, and further incubation with FITC-conjugated swine anti-rabbit immunoglobulins. Positive cells were analysed by flow cytometry.
• TF 1-2I9 /FVIIa bound equally efficient to both TFPI,. 276 -CD4 (Fig. 14A) and TFPI, .lg3 -CD4 (Fig. 14B), while no binding at all was detected to control cell line 531.
• FVIIa was inactivated by pre-incubation with 1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone, dihydrochloride ("1,5-DNS-GGACK-HCl"). This binds to the active site of FVIIa and inhibits binding to TFPI whilst not affecting the formation of the TF,. 2ig /FVIIa complex (Bajaj, 1992).
• FVIIa was first incubated with a 100-fold molar excess of 1,5-DNS-GGACK-HCl for 18 hours at 20°C and repurified by ion-exchange chromatography. Active-site inhibited FVIIa (FVIIai) was incubated with an equimolar concentration of TF,. 219 at 25°C for 15 minutes and then added to 2.5x10 5 cells in 100 ⁇ l. Subsequent steps were as described above.
• Kunitz domain I also retains its function when tethered to the cell surface in TFPI,. 183 -CD4 and TFPI,. 276 -CD4. It is therefore apparent that TFPI tethered at the cell surface is functionally active as a whole.
• TFPI-CD4 expressed on IPEC binds relevant human clotting factors and porcine TF
• the TFPI-CD4 fusion protein can be expressed on IPEC and retains the ability to bind FXa and FVIIa.
• a competitive inhibition approach using soluble human TF was adopted.
• Figure 19 A in the presence of saturating concentrations of FXa and FVIIa, the binding of soluble human TF to TFPI-transfected IPEC (pre-treated with IL-l ⁇ ) was significantly reduced compared to the binding by TF-negative control transfectants (not IL-l ⁇ activated).
• FIG. 20A shows the results of a single representative experiment to illustrate the procoagulant phenotype of TFPI-CD4-transfected IPEC.
• the presence of the fusion protein on transfected cells consistently prolonged clotting times when compared with control IPEC. This effect was only observed, however, after IL-l ⁇ activation - TFPI-CD4 expression had no influence on clotting times when TF-negative IPEC were used.
• the TFPI-CD4 as expected, inhibited TF-dependent, but not TF-independent fibrin generation.
• This single-stranded DNA was annealed to complementary oligonucleotides to give a double-stranded molecule. Restriction sites are included at either end of the double-stranded
• DNA to which is ligated a CD4 anchor and a P-selectin signal sequence in a similar way to that described above.
• the resulting molecule was ligated, as before, into the pH ⁇ Actpr-lgpt vector.
• a snake cDNA library could be screened on the basis of the known protein sequence.
• Clarke AR The adenovirus and the egg: a new approach to transgenesis. Nature Biotech. 1996; 14: 942.
• Cytoplasmic domain of P- selectin contains the signal for sorting into the regulated secretory pathway. Molec. Biol. Cell. 1992; 3: 309-321.
• GMP-140 a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Ce//.1989; 56: 1033-44
• Kisiel E Kondo S, Smith KJ, McMuUen BA, Smith LF. Characterization of a protein C activator from Agkistrodon contortrix contortrix venom. J.Biol.Chem. 1987;262:12607-13.
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